Nash H M, Bruner S D, Schärer O D, Kawate T, Addona T A, Spooner E, Lane W S, Verdine G L
Harvard University, Department of Chemistry and Chemical Biology, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Curr Biol. 1996 Aug 1;6(8):968-80. doi: 10.1016/s0960-9822(02)00641-3.
Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins.
Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G.
S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.
活性氧、电离辐射及其他自由基生成剂会引发DNA中鸟嘌呤（G）残基向8-氧代鸟嘌呤（OG）的转化，OG具有高度致突变性，因为在复制过程中它优先与腺嘌呤（A）错配。细菌通过一个多组分系统应对这种威胁，该系统切除损伤、纠正OG:A错配并清除dOGTP核苷酸前体池。尽管生化证据表明真核生物中存在针对OG的碱基切除DNA修复蛋白，但对这些蛋白了解甚少。
利用底物模拟亲和层析，随后采用基于机制的共价捕获程序，我们从酿酒酵母中分离出一种碱基切除DNA修复蛋白，该蛋白处理与胞嘧啶相对的OG（OG:C），但对OG:A的作用较弱。利用该蛋白的肽序列搜索酵母基因组数据库，鉴定出一个基因OGG1，其编码一个预测的43 kDa（376个氨基酸）蛋白，与通过互补克隆独立鉴定出的一个蛋白相同。Ogg1具有OG:C特异性碱基切除DNA修复活性，还具有内在的β-裂解酶活性，该活性通过席夫碱中间体进行。在酵母中对OGG1基因进行靶向破坏，揭示了另一种OG糖基化酶/裂解酶蛋白，暂命名为Ogg2，它与Ogg1的不同之处在于它优先作用于OG:G。
酿酒酵母有两种OG特异性糖基化酶/裂解酶，它们对损伤对面碱基的偏好有显著差异。我们认为其中一种Ogg1在整体三维结构上与大肠杆菌内切核酸酶III（内切酶III）密切相关，内切酶III是一种作用于片段化和氧化损伤嘧啶的糖基化酶/裂解酶。我们最近表明，作用于烷基化碱基的单功能DNA糖基化酶AlkA在结构上与内切酶III同源。我们现在在这三种蛋白中鉴定出一个共享的活性位点基序。利用这个基序作为蛋白质数据库搜索工具，我们发现它存在于许多其他处理不同损伤的碱基切除DNA修复蛋白中。因此，我们提出存在一个DNA糖基化酶超家族，其成员具有共同的折叠结构，但作用于从紫外线光加合物到错配、再到烷基化或氧化碱基等显著不同的损伤。
